This proposal is in response to RFA AI- 01-004 "Infectious Etiology of Chronic Diseases: Novel Approaches to Pathogen Detection" and specifically addresses the study of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis). M. paratuberculosis is the etiological agent of Johne's disease, chronic granulomatous enteritis in cattle and other ruminants, and has been implicated as a possible cause of Crohn's disease in humans. The difficulty in confirming or refuting an etiological link between M. paratuberculosis and Crohn's disease is a reflection of two issues associated with M. paratuberculosis: 1) the very slow growth of the bacterium engenders a long incubation period in the host and hinders detection; and 2) there is a poor understanding of the biochemistry and genetics of this organism. It is our contention that the second issue can be best addressed by a molecular definition of M. paratuberculosis. This goal will be accomplished by four specific aims: 1) use standard proteomic methods and develop a new ICAT- based method to identify M. paratuberculosis-specific gene expression; 2) perform genomic analyses by using Suppression Subtractive Hybridization to identify M. paratuberculosis-specific sequences; 3) characterize the polysaccharides, lipoglycans and lipids expressed by M. paratuberculosis to provide a complete biochemical analysis that will assist in defining chemical markers for this organism; and 4) develop a proteome website and a reagent repository (including recombinant M. paratuberculosis-specific proteins and clinical isolates) as a service to other basic researchers and clinicians. We will employ the strategies that are currently used by the Mycobacteria Research Laboratories at Colorado State University to support tuberculosis and leprosy research throughout the world to provide a rapid and economic means to obtain information required to develop new diagnostics and vaccines, and elucidate the biochemical and genomic differences that allow M. paratuberculosis to maintain a specific biological niche that is not shared by the closely related M. avium subspecies avium.